Device for cultivating cells and propagating viruses

ABSTRACT

This invention relates to methods for the cultivating cells, and in particular to methods for propagating viruses.

This application is a divisional application of U.S. application Ser.No. 09/679,703, filed Oct. 5, 2000 now U.S. Pat. No. 6,783,983, which isa divisional application of U.S. application Ser. No. 09/014,444, filedon Jan. 28, 1998, now U.S. Pat. No. 6,146,891, which claims priorityfrom U.S. Provisional Application No. 60/035,540 filed on Jan. 31, 1997,all of which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Many established cell lines are available for a variety of purposes inbiotechnology. Some cell lines can be cultivated as single-cellsuspensions, but other cell lines do not grow well without a support.The growth of a cell line that requires support is often limited by thesurface area available for the cells to grow on, since many cell lineswill form only a monocellular layer on the surface. In addition, somecell lines may tend to grow in clumps or aggregates in the absence of asupport, which is an undesirable result when they are needed assingle-cell suspensions, but more especially when the cells are to beinfected with a virus or transformed with a recombinant vector, sincethe virus or vector may not gain access to the cells within the clump oraggregate. Thus, there can be severe problems in scaling up thecultivation of a cell line, in particular in providing enough surfacearea for the cells to grow on and/or avoiding clumping of the cells.

Microcarrier technology has been used to cultivate cells in culture. Forexample, Forestell et al. (Biotech. Bioeng. 40: 1039-1044 (1992))disclosed extended serial subculture of human diploid fibroblasts onmicrocarriers using a medium supplement that reduced the need of thecultured cells for serum. Furthermore, Ohlson et al. (Cytotechnology14:67-80 (1994)) disclosed the bead to bead transfer of Chinese hamsterovary cells using macroporous gelatin microcarriers. Finally, Hu et al.(Biotech. Bioeng. 27: 1466-1476 (1985)) disclosed the serial propagationof mammalian cells on microcarriers using a selection pH trypsinizationtechnique.

However, in view of the problems noted above, there is a need forimprovements in methods of cultivating cell lines, in methods ofproducing viruses for clinical uses, and, in methods of scaling up theproduction of viruses for larger-scale commercialization. The instantinvention meets these needs and more.

SUMMARY OF THE INVENTION

One aspect of the invention is a method for cultivating cells,comprising:

-   -   (a) cultivating the cells on a first batch of microcarriers        until the cells are substantially confluent;    -   (b) detaching the cells from the microcarriers without removing        the microcarriers from suspension;    -   (c) Adding a second batch of microcarriers; and    -   (d) cultivating the cells further.

Another aspect of the invention is a method of detaching cells from afirst batch of microcarriers comprises the following steps:

-   -   (a) washing the microcarriers and attached cells to remove        soluble materials;    -   (b) contacting the microcarriers and washed cells with a        chelating agent;    -   (c) removing the chelating agent;    -   (d) trypsinizing the cells for a short period to detach the        cells from the microcarriers; and    -   (e) neutralizing the trypsin by adding protein,        wherein (a)-(e) are conducted in a single cultivation vessel.

A further aspect of the invention is a method for the separation ofcells from microcarriers on which they have been cultivated but fromwhich they have become detached, comprising introducing an aqueoussuspension of cells and microcarriers through an inlet into a separationdevice, the device comprising

-   -   (a) an inlet;    -   (b) a column;    -   (c) an outlet for the collection of cells and the aqueous        solution; and    -   (d) a mesh screen;        wherein the microcarriers are retained in suspension by an        upward flow in the separation device and are retained in the        separation device by a mesh screen, and wherein the cells and        aqueous solution are collected through the outlet.

A further aspect of the invention is a system for separating cells frommicrocarriers on which the cells have been cultivated, the systemcomprising;

-   -   (a) a bioreactor in which the cells were cultivated on the        microcarriers;    -   (b) a flow path from the bioreactor to a separation device;    -   (c) a separation device comprising        -   (I) an inlet;        -   (ii) a column;        -   (iii) an outlet for the collection of cells and the aqueous            solution; and        -   (iv) a mesh screen;        -   wherein the microcarriers are retained in suspension by the            upward flow in the separation device and are retained in the            separation device by a mesh screen, and the cells and            aqueous solution are collected through the outlet; and    -   (d) a pump, wherein the pump directs the flow of the aqueous        solution from the bioreactor to the outlet.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a side view of a system according to the present inventionused for separating media containing free cells and virus frommicrocarriers.

FIG. 2 is an exploded view of a separation device according to thepresent invention used for separating media containing free cells andvirus from microcarriers.

DETAILED DESCRIPTION OF THE INVENTION

The instant invention addresses the large scale cultivation of cells forthe propagation of viruses, especially recombinant viruses for genetherapy, vaccine production, and so on. In particular, the instantinvention addresses three aspects of large scale cultivation; the use ofbead-to-bead transfer of adherent cells to sequentially scale up thenumber of cells in culture, including the use of trypsin to dissociatecells from microcarriers in bioreactors, the use of fluidized bed-likeseparation of cells from the beads during harvest, and the use ofmicrofiltration to disrupt cells so as to liberate virus particles.

The term “virus” as used herein includes not only naturally occurringviruses but also recombinant viruses, attenuated viruses, vaccinestrains, and so on. Recombinant viruses include but are not limited toviral vectors comprising a heterologous gene. In some embodiments, ahelper function(s) for replication of the viruses is provided by thehost cell, a helper virus, or a helper plasmid. Representative vectorsinclude but are not limited to those that will infect mammalian cells,especially human cells, and can be derived from viruses such asretroviruses, adenoviruses, adeno-associated viruses, herpes viruses,and avipox viruses. Adenoviral vectors are preferred. Type 2 and type 5adenoviral vectors are more preferred, with type 5 adenoviral vectorsbeing especially preferred. ACN53 is a recombinant adenovirus type 5encoding the human wild-type p53 tumor-suppressor protein and isdescribed, for example, in published PCT international patentapplication WO 95/11984.

As used herein, the term “confluent” indicates that the cells haveformed a coherent monocellular layer on the surface (e.g., of themicrocarrier), so that virtually all the available surface is used. Forexample, “confluent” has been defined (R. I. Freshney, Culture of AnimalCells—A Manual of Basic Techniques, Wiley-Liss, Inc. New York, N.Y.,1994, p. 363) as the situation where “all cells are in contact allaround their periphery with other cells and no available substrate isleft uncovered”. For purposes of the present invention, the term“substantially confluent” indicates that the cells are in generalcontact on the surface, even though interstices may remain, such thatover about 70%, preferably over about 90%, of the available surface isused. Here, “available surface” means sufficient surface area toaccommodate a cell. Thus, small interstices between adjacent cells thatcannot accommodate an additional cell do not constitute “availablesurface”.

The cultivation steps in the methods of the present invention can becarried out in a bioreactor or fermentor known in the art of about 1 to5000 L equipped with appropriate inlets for introducing the cells andmicrocarriers, sterile oxygen, various media for cultivation, etc.;outlets for removing cells, microcarriers and media; and means foragitating the culture medium in the bioreactor, preferably a spin filter(which also functions as an outlet for media). Exemplary media aredisclosed in the art; see, for example, Freshney, Culture of AnimalCells—A Manual of Basic Techniques, Wiley-Liss, Inc. New York, N.Y.,1994, pp. 82-100. The bioreactor will also have means for controllingthe temperature and preferably means for electronically monitoring andcontrolling the functions of the bioreactor.

Exemplary microcarriers on which the cells are allowed to grow are knownin the art and are preferably specially adapted for the purpose of cellcultivation. General reference is made to the handbook Microcarrier CellCulture—Principles & Methods, published by Pharmacia. However, it shouldbe noted that some cell lines used in the present invention may notadhere strongly to the surfaces of microcarriers; it is well within theability of one of ordinary skill in the art to determine a suitablecombination of cell line, virus (where applicable), microcarrier andculture conditions. The microcarrier preferably has a particle size inthe range of about 100 to 250 microns, more preferably in the range ofabout 130 to 220 microns, and should be composed of a non-toxicmaterial. The median of the sample size preferably falls in theseranges, such that these size ranges are preferably those of at least themiddle 90% of the microcarrier sample. In a preferred embodiment, themicrocarrier consists of substantially spherical microbeads with amedian particle size of about 150 to 200 microns, preferably 170 to 180microns. The microcarrier surface may be treated to modify celladhesion, in particular to enhance cell adhesion yet permitproliferation and spreading; thus the microcarriers may be coated, e.g.,with collagen. Preferably, the microcarriers are slightly denser thanthe culture medium, so that gentle agitation will keep them insuspension, whereas simple means such as sedimentation or centrifugationallows their separation. A density of 1.03 to 1.045 g/ml when themicrocarriers are equilibrated with a standard solution such as 0.9%NaCI (or with the culture medium) is suitable. The present inventorshave found that Pharmacia's CYTODEX-3 microcarriers in general will meetthese requirements, although the particular requirements that apply forcertain cell lines or viruses may require the selection of a particularCYTODEX microcarrier.

The cells may be those of any suitable host cell line that is able toreplicate itself and in particular support the replication of the virusof interest. A particularly preferred cell line is the human embryonickidney cell line 293 (ATCC catalog number CRL 1573). These cells do notadhere strongly to all microcarriers, and are preferably used withPharmacia's CYTODEX-3 microcarriers, which are collagen-coated forbetter cell adhesion. CYTODEX-3 microcarriers have a median particlesize of about 175 microns with the middle 90% of the sample having asize of about 140 to 210 microns; the density of such microcarriers whenequilibrated with 0.9% NaCl is 1.04 g/ml. The cells are preferablycultivated on such a microcarrier in a first step, and then loosenedtherefrom and transferred to additional microcarriers for a productionstep.

Stirring can conveniently be effected not only by a paddle at the bottomof the bioreactor but also by a rotating spinfilter, which preferablyextends downwards from the top of the bioreactor into the bulk of themedium. The cells and microcarriers can be kept in suspension in theculture by rotation of the spinfilter; the spinfilter may also beequipped with fine orifices that permit the removal of medium withoutloss of cells. The medium can be removed and replaced simultaneously oralternately; it is frequently convenient to remove a substantialfraction (e.g., up to about 50%) of the medium and then replenish itwith the appropriate replacement medium while still removing medium,e.g., through the spinfilter.

Typically, cells are scaled-up from a master working cell bank vialthrough various sizes of T-flasks, and, preferably, finally tobioreactors. A preferred flask is the CELL FACTORY™ tissue culture flask(CF; NUNC), a specially designed large flask that conveniently hasseveral internal compartments providing a large surface area to whichthe cells can adhere or attach and on which they can grow. Aftercultivation until substantially confluent, the cells can be loosened bytrypsinization and isolated. The trypsinization is effected for a shortperiod (preferably less than 5 minutes, more preferably about 3minutes), and the trypsin is then neutralized by the rapid addition ofserum in growth medium. If desired, the cells can be centrifuged and thetrypsin-containing medium removed before the serum is added. Theresulting cell suspension is then typically fed into a seed productionbioreactor (typically 20-30 L volume) for further cultivation, and insome embodiments, to a larger production bioreactor (typically 150-180 Lvolume).

The ratio of volume of the second (larger) bioreactor to the seedbioreactor depends upon the degree to which the cell line is propagatedin the first bioreactor, but is typically from 5:1 to 10:1, e.g., in therange of (6-8):1.

Cells are detached from microcarriers by a trypsinization procedureperformed in the cultivation vessel, preferably a bioreactor, while themicrocarriers are suspended. The spinfilter is utilized to performmedium exchanges to reduce the serum and calcium levels in the medium,which increases the efficiency of the trypsinization while maintaining aconstant volume in the bioreactor. Settling steps are avoided whichmight cause damage to the cells on the microcarriers. The resultantcell/microcarrier suspension can then be transferred to a productionbioreactor which is previously charged with culture media andmicrocarriers.

After the transfer of cell/microcarrier suspension from the seedbioreactor, the production bioreactor (for example, about 200 L) isoperated, e.g., at about 37° C. and about pH 7.3. A perfusion of freshmedium during cell propagation can then be performed in order tomaintain the lactate concentration below about 1.0 g/L. Cells aretypically allowed to grow on the microcarriers for about 4 to 7 daysuntil more than 50% of the microcarriers are completely confluent.Preferably, a virus infection process is then initiated. A vial of 40 to50 ml viral inoculum, typically containing approximately 1.0×10¹³ totalviral particles is used to infect the production bioreactor. Virus isallowed to replicate in the production bioreactor for about 3 to 5 daysuntil about the time of maximum virus titer. Typically, more than 90% ofcells will have detached from the microcarriers due to cytopathiceffects of the virus. The final recombinant adenovirus yield from theproduction bioreactor is typically about 8.5×10⁹ viral particles/ml.This gives a total yield of viral particles of 1.4×10¹⁵ from each 160-Lbatch.

In other embodiments of the invention, the production bioreactor isinoculated with cells harvested by trypsinization and then used directlyto inoculate the production bioreactor. Typically 8 to 12 CELL FACTORY™tissue culture flasks are utilized to achieve the overall bioreactorinoculum seeding density of 0.6 to 1×10⁵ cells/ml. The typical virusyield in this method ranges from about 1.7 to 2.6×10¹⁰ viralparticles/ml. Therefore, this particular method provides a total virusparticle number of about 3 to 4×10¹⁵ from each 160 L batch.

In some embodiments of the invention, a fluidized bed-like process isused to harvest the cells from the bioreactor. Typically, the bioreactoris harvested after about 90% of the cells detach from the microcarriers.Without being limited to any one theory, the cytopathic effect of viralpropagation in the host cells appears to be responsible for the celldetachment. In other embodiments, uninfected cells can be detached frommicrocarriers by the trypsinization method of the instant invention.After the bioreactor is harvested, the broth contains cells,microcarriers and medium. Virus is present in the cells and the medium.Therefore, all of this material is preferably collected for processing.The specific gravity (density) of the microcarriers is similar to thatof the cells. Preferably, the microcarriers are kept freely suspendedwhile separating the cells from the beads, as processing steps usingsedimentation causes the cells to settle with the microcarriers, whichresults in recovery losses.

A preferred embodiment of a separation device is provided in FIGS. 1 and2. In some embodiments of the invention, the separation device isprovided as part of a system. An exemplary system is depicted in FIG. 2.The system thus comprises a bioreactor 100 in which the cells arecultivated on microcarriers; a flow path 102 from the bioreactor to theseparation device 104; the separation device comprising a column 106; anoutlet 108 for the collection of cells and the aqueous solution; and amesh screen 110. The microcarriers are retained in suspension by anupward flow in the separation device and are retained in the separationdevice by the mesh screen, and the cells and aqueous solution arecollected through the outlet. Also provided in the system is a pump 112,wherein the pump directs the flow of the aqueous solution from thebioreactor to the outlet. In some embodiments, a microfilter 114 and anultrafilter 116 may be provided as components of the system.

In the embodiment shown in FIG. 1, the separation device typicallycomprises a column 106, such as a chromatography column, having an inlet114 through which an aqueous suspension of cells and microcarriers froma bioreactor 100 is introduced into the separation device 104; and atleast one outlet 108 for the collection of cells and the aqueoussolution; and a mesh screen 110. The microcarriers are retained insuspension in the column by an upward flow in the separation device andare retained in the separation device by a mesh screen, and wherein thecells and aqueous solution are collected through the outlet. The flowrate in the separation device is about 1 to about 3 cm/min. Typically,an upward flow through the column is generated by pumping an aqueoussolution, such as the cell suspension or a buffer, through the inlet,wherein the inlet is situated at the bottom of the device and the outletis situated at the top of the device.

FIG. 2 is an expanded schematic view of a separation device 200,depicting in more detail an outlet assembly 210, a mesh screen assembly212, an inlet 214, and a column 216 having an upper section 218 and alower section 220. The lower section typically comprises about 20 to50%, more preferably about 30%, of the volume of the column and containsthe inlet. The lower section is preferably conical, with a preferredangle of about 15 to about 45 degrees.

Thus, the fermentation broth from the bioreactor is pumped into the baseof the column. The flow rate is regulated to provide an upward flowsufficient to keep the cells and viral particles suspended in the mediumwhile allowing for the retention of the microcarriers within theseparation device. Preferably, the flow rate is approximately 1-2 cm/minsince the cells have a specific gravity similar to that of themicrocarriers. The clarified broth containing cells and virus passesthrough the mesh screen on the upper end of the fluidized bed-likecolumn and is collected for microfiltration.

For a 200 L scale device, the lower section of the column preferably isconical. The cone allows for a gradual reduction of the linear velocityof the fermentation broth entering the cone. The fluid velocity of theinlet line is reduced to achieve a reduced linear flow in a uniformdistribution across the cross sectional area at the upper end of thecone. The walls of the cone are at an angle which allows the beads thatsettle on the walls to move downward to the inlet. In this way, thesebeads are resuspended to avoid entrapment of the cells between thesettled beads. The angle of the conical walls is preferably about 30degrees. Angles less than 15 degrees provide an exceptionally long coneand angles greater than approximately 45 degrees may not disperse theinlet feed effectively. The upper section of the column functions as azone in which the beads settle at a rate greater than the linear flowrate of the fermentation medium. This section of the column iscylindrical in shape. Within this zone a boundary is formed such thatthe microcarriers accumulate in the lower region of the column. An endplate assembly 222 (FIG. 2) of the column functions as a collectionpoint for the clarified fermentation media containing cells and virus.This consists of an end plate 224 fitted with a mesh screen assembly.This screen, preferably about 50 to 120 mesh, more preferably about 100mesh, functions as a second point for removal of the microcarriers.

The above-described embodiment is the preferred embodiment used in theexamples herein. The column dimensions and screen mesh may be variedbased upon the volume of solution to be processed, the concentration ofbeads, the particular microcarrier used and the media formulation (e.g.,specific gravity of media). A preferred column consists of a bottom conecustom fabricated out of stainless steel to be attached to two PharmaciaKS370 section tubes fitted with a KS370 end assembly to which the vendorscreen was replaced with a stainless steel (ss) mesh (preferably about50 to 120 mesh, more preferably about 100 mesh).

After the cells are collected, they are preferably lysed to liberateadditional virus particles. Homogenization or freeze-thawing may be usedto liberate the virus particles. In a preferred embodiment of theinvention, microfiltration is used to simultaneously lysevirus-containing cells and clarify the broth of cell debris which wouldotherwise interfere with viral purification. For example, themicrofiltration can be performed using a PROSTAK (Millipore) system witha 0.65 micron, hydrophilic or hydrophobic membrane and at a shear rateof 7000 1/sec. The shear rate is generated by the flow of retentatethrough the tangential flow channels of the membrane. Therefore, thecross-flow is used not only to prevent the membrane from fouling but canalso be used to create enough shear for lysing the cells. The pore sizeof the filter should be sufficient to allow passage of virus whileretaining cell debris. Thus, typically the pore size range is about0.2-0.65 micron. The shear rate range is typically about 2000 to 10,0001/sec, more preferably about 7000 1/sec.

Typically, BENZONASE™ endonuclease (American International Chemical,Inc.) is added to the clarified broth to digest cellular nucleic acids,as viral particles can become complexed with cellular nucleic acids. Ina preferred embodiment, ultrafiltration using a PELLICON system(Millipore) with a 1 million nominal molecular weight cut-off, PELLICONI-regenerated cellulose membrane is used to concentrate the virus. Theultrafiltration step accomplishes two functions; the virus isconcentrated for purification and diafiltration is performed to exchangethe buffer so that the virus suspension can be applied directly to aDEAE column. The eluate from the microfilter contains the liberatedvirus and is preferably concentrated ag., by ultrafiltration.

Between each cultivation step, the cells can be loosened and strippedfrom the microcarrier by trypsinization, e.g., by treatment withtrypsin. In the present invention it is preferred to remove the serumused in the cultivation, since the serum proteins inhibit the trypsin;removal of the serum therefore allows a smaller amount of trypsin to beused. This is advantageous since addition of a larger amount may causelocalized high concentrations of trypsin that could damage the cells.With regard to the next step, Ca⁺⁺ ions are removed since the removal ofthese ions from the cells tends to loosen the cells and enables one touse less trypsin. Thus, loosening and stripping cells, especially of thehuman embryonic kidney cell line 293, can conveniently include thefollowing steps:

-   -   i) rapidly washing the cells to remove serum and other soluble        materials;    -   (ii) removing Ca++ from the washed cells by adding a chelating        agent;    -   (iii) rapidly removing the chelating agent;    -   (iv) rapidly adding trypsin;    -   (v) trypsinizing the cells for a short period of time        (preferably ranging from about 3 minutes to about 15 minutes);        and    -   (vi) rapidly neutralizing the trypsin by adding protein.

In step (i) above, the phrase “rapidly washing” means at a constantbioreactor volume perfusing one volume change of medium at a rate ofabout 1-3 liters per minute, more preferably about 2 liters per minute.In step (iii) above, the phrase “rapidly removing the chelating agent”means at a constant bioreactor volume perfusing one and a half volumechanges of medium at a rate of about 1-3 liters per minute, morepreferably about 2 liters per minute. In step (iv) above, the phrase“rapidly adding trypsin” means adding the appropriate volume of trypsinsolution (typically a 2.5% solution) at a rate of about 1-3 liters perminute, more preferably about 2 liters per minute. In step (vi) abovethe phrase “rapidly neutralizing the trypsin by adding serum” meansadding the appropriate volume of serum at a rate of about 1-3 liters perminute, more preferably about 2 liters per minute.

If the serum is not removed in step (i), then the addition of thenecessary large amounts of trypsin can lead to locally highconcentrations of trypsin, which can actually damage or even kill thecells rather than simply loosen them. The removal of serum in Step (i)and of Ca⁺⁺ in Step (ii) reduces the amount of trypsin needed in Steps(iv) and (v). To avoid actually damaging or even killing the cells, thetreatment with the chelating agent and with the trypsin shouldpreferably be kept short (i.e., long enough to detach the cells from themicrocarriers, but preferably not longer). Examples of preferredchelating agents include EDTA (ethylene diamine tetraacetic acid) andEGTA (ethylene-bis(oxyethylene-nitrilo)tetraacetic acid).

The serum is removed by a process of medium exchange; for example, themedium can be pumped off through a spinfilter. Serum-free wash medium isadded to replace what was pumped off and the mixture stirred.Alternatively, the addition of serum-free wash medium can be continuouswith the removal of medium through the spin-filter. The process isrepeated until the serum concentration has been reduced to asufficiently low level, e.g., less than about 1.0 to 0.2%, preferablyabout 0.2%. The chelating agent, preferably EDTA, is added in serum-freechelating medium, the mixture again stirred, and the chelating agentpumped off. Alternatively, the addition of the chelating agent inserum-free medium can be continuous with the removal of the mediumthrough the spinfilter.

The trypsin is preferably used in step (v) to provide a concentration inthe bioreactor of from about 0.05 to 0.1%, and it is allowed to act onthe cells for from 5 to 10 minutes, e.g., preferably a trypsinconcentration of about 0.065% for about 8 minutes. Protein, typically inthe form of bovine calf serum, is preferably added to the bioreactor ina final concentration of about 10 to 20% to inhibit the trypsin.

Thus the addition of serum in step (vi) not only prepares the cells forfurther cultivation but also neutralizes residual trypsin. The entiresequence of steps (i)-(vi) can take place in situ in the bioreactor; insome embodiments the suspension of microcarriers and cells can betransferred to a larger bioreactor, where further microcarriers areadded for the next step of the cultivation. The cells are allowed toattach to the microcarriers and then are cultivated further. Once theybecome substantially confluent again (e.g., 3-4 days' cultivation atabout 37° C.), they can be put through the next stage, which may forexample be harvesting, loosening for a further upstaging, or inoculationwith virus. If the cells have been cultivated simply for harvest, thenthey can be harvested at this stage, e.g., by a repetition of Steps (i)through (vi) above. If they are needed for a further upstaging, thenSteps (i)-(vi) above can be repeated. If they have been cultivated forpropagation of a virus, the virus can now be inoculated into the medium.

The examples herein serve to illustrate but do not in any way limit thepresent invention. The selected vectors and hosts and other materials,the concentration of reagents, the temperatures, and the values of othervariables are only to exemplify the application of the present inventionand are not to be considered limitations thereof.

EXPERIMENTAL EXAMPLES I. Overview

A. Cell Inoculum Preparation

Each viral fermentation batch is started from a cell line propagatedfrom a vial of 293 cell Manufacturers Working Cell Bank (MWCB). Thebioreactors are inoculated with 293 cells (ATCC catalog number CRL 1573)maintained by propagation in T-flasks and CELL FACTORY™ using growthmedium (Medium 1), as illustrated in Table 1 below. Each transferrepresents a passage. Typically, passage numbers of 4 to 30 are employedfor inoculation of a seed bioreactor.

TABLE 1 MEDIA COMPOSITION Typical Component Typical Component MediumPurpose Function Component Range Medium 1 Cell Cell Growth DMEM Powder¹10-20 g/l (Growth Growth Cell Growth Glutamine 0.1-1.0 g/l medium) CellGrowth Bovine Calf 5-15% Serum³ Buffer Sodium 2-4 g/l Bicarbonate Medium2 Prepara- Reduce Ca⁺⁺ DMEM Powder, 10-20 g/l (Serum- tion for for tryp-Ca⁺⁺ free free trypsin- sinization, Glutamine 0.1-1.0 g/l wash izationLater wash Sodium 2-4 g/l medium) EDTA Bicarbonate Cell Viability BufferMedium 3 Prepara- Reduce Ca⁺⁺ DMEM Powder, 10-20 g/l (Serum- tion forfor tryp- Ca⁺⁺ free² free trypsin- sinization chelating ization Ca⁺⁺EDTA⁴ 200-400 mg/l medium) Chelation Cell Glutamine⁵ 0.1-1.0 g/lViability Buffer Sodium 2-4 g/l Bicarbonate ¹DMEM powder (available fromAmerican Biorganics, Catalog no. D2807): preferably used to provide 4.5g/L glucose and 0.584 g/L L-glutamine; no sodium bicarbonate and noHEPES. ²Ca⁺⁺-free DMEM powder (available from American Biorganics,Catalog no. D2807403): preferably used to provide 4.5 g/L glucose and0.584 g/L L-glutamine; no sodium bicarbonate, no calcium chloride and noHEPES. ³Bovine calf serum: available from Hyclone, Catalog no. 2151.⁴EDTA stock solution: 186.1 g/L EDTA and 20 g/L NaOH pellets. ⁵Glutaminestock solution: 29.22 g/L.

To prepare for the transfer of cells from flask to flask during cellexpansion, the spent medium is poured off and the cells in the flask arethen washed with phosphate-buffered saline (PBS). A trypsin solution isadded to the cell monolayer on the flask's surface and the cells areexposed until they detach from the surface. Trypsin action is thenlargely neutralized by adding growth medium (Medium 1, Table 1)containing serum; complete neutralization is not necessary, sinceresidual trypsin will have low activity. The cells can be recovered bycentrifugation and resuspended in fresh growth medium (Medium 1). Table2 shows the typical volumes are used.

TABLE 2 TYPICAL VOLUMES USED IN TRANSFER OF CELLS Volume of serum-Volume of containing Volume of 0.05% Medium to Medium Flask SurfaceTrypsin Inactivate for Cell volume area Solution Trypsin Growth T-75 75cm² 3 ml 10 ml 30 ml T-500 500 cm²  25 ml  50 ml 200 ml  CELL 6000 cm² 250 ml  500 ml  1500 ml  FACTORY ™B. Virus Inoculum Preparation

Virus inocula can be prepared by infecting mature CELL FACTORY™ tissueculture flasks. In this procedure, 293 cells are first propagated fromT-flasks to CELL FACTORY™ tissue culture flasks. When CELL FACTORY™tissue culture flask cultures are mature (typically 80-90% confluent)they are infected with an inoculum from the manufacturer's working virusbank (MWVB). The infected CELL FACTORY™ tissue culture flasks areincubated until the 293 cells detach from their supporting surface. Thecells are collected by centrifugation and ruptured by multiplefreeze-thaw cycles. After a subsequent centrifugation, the virus isrecovered in the supernatant and stored as aliquots at −20° C. or below.This material is the “Virus Inoculum” which is used to infectbioreactors. Optionally, the virus inoculum can also be derived from thebioreactor harvest which is filter-sterilized (see “ProductionBioreactor Harvest” below).

C. Seed Bioreactor Preparation and Operation

Preferably, a seed bioreactor is used to prepare the 293 cell inoculumfor the production bioreactor. The seed bioreactor is steam-sterilizedand charged with a batch of filter-sterilized growth medium (Medium 1,Table 1, above) for the free cell suspension process. For themicrocarrier process, however, swelled sterile microcarrier beads(CYTODEX-3 or equivalent) are preferably added at this stage.

The seed bioreactor is inoculated with the 293 cells harvested from CELLFACTORY™ tissue culture flasks. The operating conditions are set asshown in Table 3. The pH and dissolved oxygen (DO) are controlled bysparging CO₂ and oxygen, respectively. Extra growth medium can be addedto the bioreactor by perfusion. Cell growth is monitored by microscopicexamination and by measuring the lactate production and glucoseconsumption. Typically, when the cell density in the suspension culturereaches 1×10⁶ cells/ml in the seed bioreactor, it is ready forinoculating the production bioreactor. However, inoculation requires afew additional steps for the microcarrier process. Typically, when thecells on the microcarriers are >50% confluent, the serum and calcium inthe bioreactor medium are washed off using media described in Table 1.Trypsin is then added rapidly, and when the cell detachment reachestypical level, serum is added to inactivate the trypsin. The seedbioreactor contents are now transferred to the production bioreactor.

Optionally, 293 cells harvested from multiple CELL FACTORY™ tissueculture flasks can directly be used as inoculum for a productionbioreactor. Typically, 8-12 such cultures are harvested and pooled toprovide an inoculum.

TABLE 3 SEED BIOREACTOR OPERATING CONDITIONS Variable Recommended RangeTypical or preferred Temperature 34° C.-39° C. 37° C. pH 6.9-7.5 7.3Dissolved >10% 30% air saturation Oxygen Pressure >0.05 bar 0.1 barAeration >50 Liters/hour 180 Liters/hour overlay Agitation >20 rpmInitially 50-70 rpm, with optional stepwise increases Spinfilter >20 rpmInitially 50-70 rpm, with optional stepwise increasesTrypsinization >25% of microcarriers >50% of microcarriers haveconfluent cells have confluent cellsD. Production Bioreactor Preparation and Operation

The viral production process is exemplified in a 200-L productionbioreactor using growth medium (Medium 1, Table 1). In the suspensionculture process, filter-sterilized medium is batched into thebioreactor. However, in the microcarrier process, microcarriers areeither sterilized in situ in the production bioreactor or autoclavedexternally and charged. These microcarriers are then conditioned in thegrowth medium (Medium 1) prior to inoculation with 293 cells.

The production bioreactor is inoculated with the 293 cells from the seedbioreactor. The operating conditions are set as shown in Table 4. The pHand dissolved oxygen (DO) are controlled by sparging CO₂ and oxygen,respectively. Optionally, extra growth medium can be added to thebioreactor by perfusion. Cell growth is monitored by microscopicexamination, and by measurement of lactate production and glucoseconsumption. Cells are allowed to grow to approximately 1×10⁶ cells/ml.The bioreactor is then inoculated with virus. Preferably, a multiplicityof infection (MOI) ratio expressed as the total viral particles per cellof 50:1 to 150:1 is used. Viral titer is typically performed using theResource Q HPLC assay. Virus is allowed to propagate until the cellviability drops to about 10%.

The type of virus and its action upon the host cells may determinewhether it is necessary to detach the host cells from the microcarriersand/or lyse the cells. At about the time of maximum virus titer(frequently when the cells start to detach from the microcarrier andsome of which may lyse, so that the virus starts to escape), theincubation can be stopped and the cells and virus can be harvested.Indeed, in the microcarrier process, 80-90% of the cells, sometimes evenmore than 90%, may detach from the microcarriers. Without being limitedto any one theory, the cytopathic effect of viral propagation in thehost cells appears to be responsible for cell detachment. Thus, whenadenovirus ACN53 is used with 293 cells, the cells start to detach fromthe microcarriers after 3 or 4 days' cultivation with the virus.

TABLE 4 PRODUCTION BIOREACTOR OPERATING CONDITIONS Variable RecommendedRange Typical or preferred Temperature 34 ° C.-39° C. 37 ° C. pH 6.8-7.57.3 6.8-7.6 7.4 after virus infection Dissolved >10% 30% air saturationOxygen Pressure >0.05 bar 0.1 bar Aeration >500 Liters/hour 2500Liters/hour Overlay Agitation >20 rpm 50-70 rpm Spinfilter >20 rpm 50-70rpm Virus infection >25% of microcarriers >50% of microcarriers timehave confluent cells have confluent cells Harvest time >50% ofmicrocarriers >90% of microcarriers are empty are emptyE. Production Bioreactor Harvest

1. Separation of Cells from Microcarrier

At harvest, the bioreactor contents have to be handled differentlydepending on whether the process uses free suspension or a microcarrier.In the microcarrier process, a fluidized bed column is preferably usedto separate the microcarriers from cells and supernatant. An upward flowrate is maintained so as to retain the microcarriers while the cells andsupernatant pass through. The fluidized bed is washed with medium orwash buffer to recover most residual cells and virus, and the washingsare combined with the cells and supernatant as an eluate. The fluidizedbed operation is not required for the free cell suspension process.

The eluate containing cells and virus is further processed in that thecells are lysed by high shear to release virus, and the eluate is thenclarified by means of a cross-flow microfiltration. Typically, 0.65 μmDURAPORE (Millipore) or equivalent membranes are used. Towards the endof the microfiltration, the retentate is washed with wash buffer torecover the residual virus into the permeate. After microfiltration, thepermeate can be optionally treated with a nuclease such as BENZONASE™endonuclease.

The permeate from the microfiltration is concentrated by ultrafiltration(with a typically 1 million molecular weight cutoff) and a bufferexchange is performed using the wash buffer. The concentrated anddiafiltered retentate containing the virus is then passed through afinal filter. The resulting filtrate is stored as “Viral Concentrate” ina freezer at −20° C. or below.

2. Composition and Preparation of Media

Table 1 above lists the media used in the preparation of the viralinoculum and in the fermentation process. All these culture media areprepared by first dissolving the dry DMEM powder and other reagents inpurified water. After dissolving the dry powders, these media areadjusted to pH 7.2-7.6 with hydrochloric acid. The media are thensterilized by passing through a 0.2 μm filter into an appropriatestorage container. The sterile media are refrigerated below 10° C. anddiscarded one month after preparation.

Table 5 lists various buffers used in the process.

TABLE 5 BUFFERS USED IN FERMENTATION AND HARVESTING Typical ComponentTypical Component Step Procedure Function Component Range Cell Wash withCell Potassium 0.1-0.3 g/l inoculum PBS prior Electrolyte Chlorideprepara- to tryp- Balance tion in sinization Cell Potassium 0.1-0.3 g/lflasks Electrolyte Phosphate, Balance monobasic Ionic Sodium 6-10 g/lStrength Chloride Buffer Sodium 0.5-2 g/l Phosphate, dibasic BufferSodium 1-3 g/l Phosphate, dibasic heptahy- drate Microcar- Swell SameSame Same ranges rier prep- micro- functions as components as above +aration carriers above + as above + 0.1-1 ml/l with Wetting agent Tween80 PBS and Tween 80 Produc- Wash fluid- Buffer Tris Base 4-8 g/l tionbio- ized bed, Ionic Sodium 7-11 g/l reactor microfilter StrengthChloride harvest and ultra- Stabilizer Magnesium 0.2-0.6 g/l filterChloride retentate Stabilizer Sucrose 16-24 g/l with Tris Buffer

Table 6 summarizes the in-process controls during the fermentation andharvesting processes.

TABLE 6 IN-PROCESS CONTROLS Step In-Process Control Recommended RangeT-flasks; CELL Microscopic Normal growth and FACTORY ™: observationmorphology. No Preparation of contaminants. inoculum Seed bioreactorMicroscope Normal cell growth observation, and morphology on measurementof microcarriers, lactate and glucose lactate <1.2 g/L atconcentrations, transfer point, no supernatant cell contaminants. count,sample sterility check. Production Microscope Normal cell growthbioreactor observation, and morphology on measurement of microcarriers,lactate and glucose lactate <1.2 g/L concentrations, prior to infection,supernatant cell majority of cells count, sample detach from sterilitycheck. microcarriers at harvest, no contaminants. Fluidized bed Visualobservation of Fluidized bed fluidized bed, feed volume to be pressureand feed smaller than total flow rate. column volume and feed pressureshould be below the leak point of the column assembly. MicrofiltrationObservation and Feed flow rate control of feed flow, adjusted to achievepermeate flow, feed, high shear without retentate and exceeding atransmembrane transmembrane pressure, retentate pressure of 0.5temperature. bar. Temperature should not exceed 37° C. UltrafiltrationObservation and Feed pressure control of feed should not exceed 1pressure and bar. Temperature retentate should not exceed temperature.37° C. Final filtration Observation and Feed pressure control of feedshould not exceed 2 pressure bar

II. Bead to Bead Transfer from Seed Bioreactor to Production Bioreactor

A. 293 Cell Inoculum Preparation for Seed Bioreactor

1. Scale-up from Vial to T75 Flask

A frozen vial of the 293 cell line containing a total of 2×10⁷ cells wasthawed in a 37° C. water bath. The cells were washed with 10 ml ofMedium 1. The washed cells were resuspended in a total volume of 30 mlof Medium 1 and placed in a 75 cm² tissue culture flask (T75). Theculture was placed in an incubator at 37° C. with a 5% CO₂ atmosphereand a humidity level of 100%. This was passage 1 of the culture.

2. Scale-up from T75 Culture to T500 Flask Using Trypsinization

The T75 culture reached a confluency level of 90% in three days. At thistime the T75 culture was trypsinized in the following manner. The 30 mlof supernatant medium was removed from the flask. A volume of 10 ml ofCMF-PBS (Dulbecco's phosphate buffered saline without calcium chlorideand without magnesium chloride) was used to wash the culture surface.The supernatant CMF-PBS was removed from the flask. Two ml of TE (0.05%crude trypsin with 0.53 mM EDTA-4Na) solution was added to the flask.The flask was moved so that the solution covered the entire culturesurface. The cells detached from the flask surface within five minutes.Ten ml of Medium 1 was added to the flask immediately after the cellsdetached from the surface. The cell suspension was centrifuged at 1000rpm for ten minutes at ambient temperature with the break. Thesupernatant was removed. The cells were resuspended in five ml ofMedium 1. The cell suspension was transferred to a sterile bottle with200 ml of Medium 1. The 200 ml cell suspension was placed in a 500 cm²tissue culture flask (T500). The liquid in the T500 was allowed toequilibrate between chambers before placement in a horizontal positionin the incubator. The culture was placed in an incubator at 37° C. witha 5% CO₂ atmosphere and a humidity level of 100%. This was passage 2 ofthe culture.

3. Scale-up and Passaging of T500 Culture Using Trypsinization

The T500 culture reached a confluency level of 90% in four days. On thefourth day, the T500 culture was trypsinized and scaled-up in thefollowing manner. The supernatant medium was discarded. The culturesurface was washed with 25 ml of CMF-PBS. A volume of 25 ml of TE wasadded to the flask. The flask was moved so that the TE solution coveredall three layers of culture surface. The cells detached from the flasksurface within five minutes. After the cells detached, 50 ml of Medium 1was added to the flask. All of the surfaces were contacted with themedium by moving the flask. The resultant cell suspension was pouredinto a 200 ml conical centrifuge bottle. The cells were pelleted bycentrifugation at 1000 rpm for ten minutes at ambient temperature withthe brake. The supernatant was discarded. The cells were resuspended in5 to 15 ml of Medium 1. The cell suspension was placed in 800 ml (200 mlper new T500 flask) of Medium 1. The cell suspension was mixed. A volumeof 200 ml of the cell suspension was added to each of four T500 flasks.The liquid level in each flask was allowed to equilibrate betweenchambers before placement in a horizontal position in the incubator. Thesplit ratio for this passage was 1:4. This was passage 3. The culturewas passaged in this manner for passages 4 through 13. At passage 14,four T500 cultures were trypsinized in the manner given above. The cellsuspensions were pooled and placed in a bottle containing 1.5 liters ofMedium 1. This cell suspension was added to a 6000 cm² CELL FACTORY™(CF) tissue culture flask. The liquid level in the CF was allowed toequilibrate between chambers before placement in a horizontal positionin the incubator.

4. Scale-up and Passaging of CELL FACTORY™ Tissue Culture Flask Cultures

The CF culture reached an 80% confluency level in three days. Thetrypsinization was performed in the following manner for passage 15. The1.5 liters of Medium 1 was drained from the CF culture. The culturesurfaces were washed with 500 (+/−100) ml of CMF-PBS. After the wash,250 (+/−50) ml of TE solution was added to the CF culture. The CF wasmoved so that the TE solution covered each of the surfaces. After thecells detached from the surface, 500 ml of Medium 1 was added to the CF.The CF was moved so that the Medium 1 contacted each of the surfaces.The resultant cell suspension was aliquotted into four, 250 ml conicalcentrifuge bottles. The cells were pelleted by centrifugation at 1000rpm for ten minutes with the brake on. The supernatant medium wasdiscarded from each centrifuge bottle. In each centrifuge bottle, thecells were resuspended in 5 ml of Medium 1. The cells suspensions werepooled into one centrifuge bottle. The three remaining centrifugebottles were washed with an additional 5-10 ml of Medium 1 which wasadded to the pooled cell suspension. This cell suspension was splitequally among six bottles containing 1.5 liters of Medium 1. Each of thesix 1.5 liter cell suspensions was added to a CF. The liquid level ofeach CF was allowed to equilibrate among the chambers before the CF wasplaced in a horizontal position in the incubator. The culture waspassaged in the same manner for passage 16.

Passaging data are provided in Table 7.

TABLE 7 PASSAGING DATA Confluency Culture Level at Source RecipientPassage Time (Days) Passage Flask Flask 1 3 not vial T75 applicable 2 390-95% 1 × T75 1 × T500 3 4 90-95% 1 × T500 4 × T500 4 3 70% 1 × T500 4× T500 5 4 90% 1 × T500 6 × T500 6 4 85% 1 × T500 8 × T500 7 3 70% 1 ×T500 4 × T500 8 4 80% 1 × T500 4 × T500 9 4 95% 1 × T500 4 × T500 10 395% 1 × T500 6 × T500 11 3 70% 1 × T500 6 × T500 12 4 90% 1 × T500 6 ×T500 13 4 70% 1 × T500 6 × T500 14 4 90% 4 × T500 1 CF 15 3 80% 1 CF 6 ×CF 16 5 80% 1 CF 6 × CF

5. Preparation of Cell Inoculum from CELL FACTORY™ Tissue Culture FlaskCultures to the Seed Bioreactor

The CF cultures reached an 80% confluency level in five days. Four ofthe six CF cultures were used to inoculate the seed bioreactor asfollows. The 1.5 liters of Medium 1 was drained from the CF culture. Theculture surfaces were washed with 500 (+/−100) ml of CMF-PBS. After thewash, 250 (+/−50) ml of TE solution was added to the CF culture. The CFwas moved so that the TE solution covered each of the surfaces.Immediately after the cells detached from the surface 500 ml of Medium 1was added to the CF. The CF was moved so that the Medium 1 contactedeach of the surfaces. The resultant cell suspension was aliquotted intofour, 250 ml conical centrifuge bottles. The cells were pelleted bycentrifugation at 1000 rpm for ten minutes at ambient temperature withthe brake on. The supernatant medium was discarded from each centrifugebottle. In each centrifuge bottle, the cells were resuspended in 5 ml ofMedium 1. The cells suspensions were pooled into one centrifuge bottle.The remaining three centrifuge bottles were washed with an additional5-10 ml of Medium 1 which was added to the pooled cell suspension. Thecell suspensions from each of the four centrifuge bottles were thenpooled together, yielding a total volume of 50-100 ml. An additionalvolume of Medium 1 was added to bring the total volume to 1000 ml. Thiswas the cell inoculum. The total amount of cells in the cell inoculumwas 2.88×10⁹ total cells and 2.84×10⁹ viable cells. The 1000 ml of cellinoculum was transferred to a sterile Erlenmeyer flask and inoculatedinto the 30 liter seed bioreactor which contained a total volume of 18liters of Medium 1 with 66 g of CYTODEX-3 microcarriers.

B. Seed Bioreactor

1. Preparation of CYTODEX-3 Microcarriers for the 30 L Seed Bioreactor

One batch of 66 grams of CYTODEX-3 microcarriers was prepared in thefollowing manner. The 66 grams of CYTODEX-3 microcarriers was placed ina five liter, glass, Erlenmeyer flask. Two liters of CMF-PBS with 0.2 mlof Tween 80 was added. The microcarriers were allowed to swell atambient temperature for five hours and thirty minutes. After thisswelling period, the supernatant CMF-PBS was decanted from the flaskleaving behind the CYTODEX-3 microcarrier slurry. The CYTODEX-3microcarrier slurry was washed with two liters of CMF-PBS thenresuspended in CMF-PBS to a total volume of two liters. The batch ofCYTODEX-3 was autoclaved in the five liter flask at 121° C. for threeand a half hours on a liquids cycle. The sterilized CYTODEX-3 batch wasused the next day for the 30 L Seed Bioreactor.

On the day of the CYTODEX-3 addition to the 30 L Seed Bioreactor thefollowing actions were performed. The supernatant CMF-PBS was decantedfrom the five liter flask. The microcarrier slurry was washed with twoliters of Medium 1. After the wash, Medium 1 was added to the flask to afinal volume of two liters.

2. Preparation of the 30 L Seed Bioreactor

The 30 L Seed Bioreactor which contained a spinfilter was cleaned andsteam sanitized. The bioreactor was sterilized for fifty minutes at 121°C. One day prior to 293 cell inoculation, the 30 L Seed Bioreactor wasfilled with 18 liters of Medium 1. The two liters containing 66 grams ofthe CYTODEX-3 microcarriers with Medium 1 solution was added to the 30 LBioreactor. The 30 L Seed Bioreactor operating conditions are listed inTable 8.

TABLE 8 30 L SEED BIOREACTOR OPERATING CONDITIONS Operating ConditionValue Value Range pH 7.3 7.1-7.4 Temperature 37° C. 35-38° C. DissolvedOxygen 30% 20-140% Bioreactor Pressure 0.1 bar 0.05-0.4 bar AerationOverlay 180 lph 100-300 lph Agitation 70 rpm 10-100 rpm Spinfilter 70rpm 10-100 rpm

3. Cultivation of 293 Cells in the 30 L Seed Bioreactor

The 293 cells were propagated on the CYTODEX-3 microcarriers for fivedays. The actual operating conditions in the 30 L Seed Bioreactor duringthis time period are listed in Table 9.

TABLE 9 OPERATING CONDITIONS IN THE 30 L SEED BIOREACTOR ActualOperating Condition Range Target Temperature 37-37.1° C. 37.° C. pH7.2-7.6 7.3 Dissolved Oxygen 28-100% 30% Pressure 0.1 bar 0.1 barAeration Overlay 200 lph 200 lph Agitation 67-94 rpm 67-94 rpmSpinfilter 69-87 rpm 69-87 rpm

On the fifth day of cultivation, 54% of the microcarrier populationcontained greater than 50 cells/microcarrier, 38% contained 1-25 cells,8% contained no cells, and 8% were in aggregates of two microcarriers asdetermined by examination of a sample under the microscope at 100×magnification. Results are provided in Table 10.

TABLE 10 RESULTS FROM MICROSCOPIC EXAM ON THE FIFTH DAY OF CULTIVATIONOF 293 CELLS ON CYTODEX 3 MICROCARRIERS IN THE 30 L BIOREACTORPercentage of Total Microcarrier Population >50 10-50 1-10 cells/ cells/cells/ empty microcarrier microcarrier microcarrier microcarrier 54% 16%17% 7%B. Bead to Bead Transfer Procedure

1. Preparation of CYTODEX-3 microcarriers for the Production Bioreactor

One batch of 420 grams of CYTODEX-3 microcarriers was prepared in thefollowing manner. The 420 grams of CYTODEX-3 microcarriers was placed ina fifty liter carboy. A volume of 21.5 liters of CMF-PBS with 2.0 ml ofTween 80 was added. The microcarriers were allowed to swell at ambienttemperature for 17 hours. After this swelling period, the supernatantCMF-PBS was removed from the carboy leaving behind the CYTODEX-3microcarrier slurry. The CYTODEX-3 microcarrier slurry was washed with25 liters of CMF-PBS then resuspended in CMF-PBS to a total volume of 20liters.

The 200 L bioreactor which contained a spinfilter was cleaned and steamsanitized. The 20 liter CYTODEX-3 microcarrier slurry was transferred tothe bioreactor. Five liters of CMF-PBS was used to wash out the 50 litercarboy and transferred to the bioreactor. The bioreactor was sterilizedfor 50 minutes at 123° C. The bioreactor was maintained at 4° C.overnight. The next day, 120 liters of Medium 1 was added to the 200 Lbioreactor. The microcarrier solution was agitated at 90 rpm for tenminutes in the bioreactor. The volume was brought down to 55 liters bywithdrawing liquid through the spinfilter. An additional 110 liters ofmedium 1 was added to the 200 L bioreactor. The microcarrier solutionwas agitated at 90 rpm for ten minutes in the bioreactor. The volume wasbrought down to 55 liters by withdrawing liquid through the spinfilter.Medium 1 was added to the bioreactor to bring the volume to 125 liters.The bioreactors operating conditions were set according to Table 11.

TABLE 11 BIOREACTOR OPERATING CONDITIONS Operating Condition TargetActual Range Temperature 37-37.1° C. 36-38° C. pH 7.3 7.1-7.4 DissolvedOxygen 30% 20-100% Pressure 0.1 bar 0.05-0.5 bar Aeration Overlay 2500lph 2500 lph Agitation 50-90 rpm 50-90 rpm Spinfilter 60-100 rpm 60-100rpm

The 200 L production bioreactor was ready to receive the inoculum fromthe seed bioreactor.

2. Trypsinization of the Seed Bioreactor Culture

On the fifth day of cultivation of the 293 cells on the CYTODEX-3microcarriers, the bead to bead transfer procedure was performed. Theserum and calcium levels of the medium in the culture were reduced byperfusing 22 liters of Medium 2 at a rate of 2 liters per minute usingthe spinfilter with a constant bioreactor volume of 20 liters. Perfusionwas continued with 22 liters of Medium 3 at a perfusion rate of 2 litersper minute with a constant bioreactor volume of 20 liters. This reducedthe serum and calcium levels further. Medium 3 contained disodiumethylenediaminetetraacetate dihydrate (EDTA) which chelates divalentcations such as magnesium and calcium. A third round of perfusion wasperformed using 33 liters of Medium 2 which was designed to furtherreduce the serum and calcium levels and to reduce the concentration ofEDTA in the medium. At this point, the medium was withdrawn through thespinfilter to reduce the total culture volume to 15.5 liters. A volumeof 480 ml of a 2.5% trypsin solution was added to the bioreactor in oneminute. By microscopic observation, eight minutes after the addition ofthe trypsin solution, 90% of the cells had detached from themicrocarriers. At this point, four liters of serum was added to thebioreactor in two and a half minutes to inhibit the action of trypsinand to protect the cells from shear during the transfer procedure to theProduction Bioreactor. The trypsinized cells and microcarriers weretransferred to the Production Bioreactor by pressure. The transfer wasachieved in eight minutes. Immediately after the transfer, five litersof Medium 1 was added to the Seed Bioreactor as a flush and transferredto the Production Bioreactor by pressure. Operating conditions of theseed bioreactor during the bead-bead transfer procedure are provided intable 12.

TABLE 12 OPERATING CONDITIONS OF THE SEED BIOREACTOR DURING THEBEAD-BEAD TRANSFER PROCEDURE Operating Condition Actual Range TargetTemperature 37° C. 37° C. pH 7.2-7.5 7.3 Dissolved Oxygen 28-55% 30%Pressure 0.1 bar 0.1 bar Aeration Overlay 200 lph 200 lph Agitation89-94 rpm 89-94 rpm Spinfilter 86-87 rpm 86-87 rpmC. Cultivation of 293 Cells in the 200 L Production Bioreactor BeforeInfection

The 293 cells were propagated on the CYTODEX-3 microcarriers for sixdays. The actual operating conditions in the 200 L Production Bioreactorduring this time period are listed in Table 13.

TABLE 13 OPERATING CONDITIONS IN THE 200 L PRODUCTION BIOREACTOROperating Condition Actual Range Target Temperature 37-37.8° C. 37° C.pH 7.2-7.5 7.3 Dissolved Oxygen 43-160% 30% Pressure 0.08 bar 0.1 barAeration Overlay 2500 lph 2500 lph Agitation 69-74 rpm 69-74 rpmSpinfilter 71-80 rpm 71-80 rpm

A total volume of 115 liters of Medium 1 was perfused from days fourthrough six. The rates were as follows; 24 liters was perfused in onehour on day four, 40 liters was perfused in one hour on day five, and 50liters was perfused in one hour on day six. The oxygen uptake ratemeasured as the decrease of the dissolved oxygen level (percent of airsaturation, % DO) per minute (% DO decrease/min) reached 1.65%/mm on daysix. Results from microscopic exam on the sixth day of cultivation of293 cells on CYTODEX-3 microcarriers in the 200 L bioreactor areprovided in Table 14.

TABLE 14 RESULTS FROM MICROSCOPIC EXAM ON THE SIXTH DAY OF CULTIVATIONOF 293 CELLS ON CYTODEX 3 MICROCARRIERS IN THE 200 L BIOREACTORPercentage of Total Microcarrier Population >50 10-50 1-10 cells/ cells/cells/ empty microcarrier microcarrier microcarrier microcarriers 31%23% 25% 21%D. Infection of the 293 Cells in the 200 L Production Bioreactor

The bioreactor culture was inoculated with virus on day 6. The viralinoculum had been stored frozen at −80° C. A volume of 45 ml of theviral inoculum, 2-2, was thawed in a water bath at 20-25° C. The totalamount of virus added to the tank was 1.1×10¹³ viral particles asmeasured by the Resource Q HPLC assay. The viral inoculum was mixed andplaced in a bottle with one liter of Medium 4 (293-1-R07 Dulbecco'smodified Eagle's medium with L-glutamine and sodium bicarbonate (3.7g/l)). The viral solution was filtered through a Gelman Maxi CultureCapsule into a sterile five liter addition flask. The viral suspensionwas added to the 200 L Production Bioreactor with sterile connectionsmade via the tubing welder. Production bioreactor operating conditionsafter infection are provided in Table 15.

TABLE 15 PRODUCTION BIOREACTOR OPERATING CONDITIONS AFTER INFECTIONOperating Condition Actual Range Target Temperature 37-37.8° C. 37° C.pH 7.08-7.23 7.4 Dissolved Oxygen 20-73% 30% Pressure 0.08 bar 0.1 barAeration Overlay 2500 lph 2500 lph Agitation 69-74 rpm 69-74 rpmSpinfilter 71-80 rpm 71-80 rpm

Three days after infection, 89% of the microcarriers did not haveattached cells and the oxygen uptake rate measured was 0.53%/min. Thetotal infected cell concentration present in the supernatant broth was1.0×10⁶ cells/ml. The total volume in the bioreactor was 162 liters. Thebioreactor was harvested at this time.

E. Recovery Operations

A volume of 400 liters of the harvest recovery buffer was prepared andfiltered through a PALL ULTIPOR N66 (0.2 micron pore size) andaliquotted into sterile vessels in the following manner. Three aliquotsof 210 liters, 130 liters, and 50 liters were prepared. The volume of210 liters was used for the bioreactor wash and the fluidized bed columnoperation. The 130 liter aliquot was used during the microfiltration.The 50 liter aliquot was utilized during the ultrafiltration process.

F. Separation of Cells from the Microcarriers using the Fluidized BedColumn

The fluidized bed was sanitized using a caustic solution (0.1 N sodiumhydroxide). A T-fitting was connected to the harvest port of thebioreactor. On one side of the T-fitting, a sanitary hose (15.9 mm id)and valve were connected to the fluidized bed column. A peristaltic pumpwas placed on this line (Watson Marlow Model 604S). The second side ofthe T-fitting was connected to a sanitary hose (15.9 mm id) and valveleading to the buffer tank. The outlet of the fluidized bed column,through which the broth containing cells and virus passed, was connectedto a tank used as the microfiltration recirculation vessel.

The broth from the bioreactor was passed through the fluidized bedcolumn at a target flow rate of 2-3 liters per minute. The flow rate wascontrolled with the peristaltic pump. Agitation was maintained in thebioreactor. When the bioreactor volume was less than 100 liters the spinfilter was turned off. When the bioreactor volume was less than 30liters, the agitator was turned off. After the bioreactor contents wereprocessed through the fluidized bed column, the bioreactor was washedwith 90 liters of harvest recovery buffer. This wash material wasprocessed through the fluidized bed column. At the end of the process,the microcarriers remained in the fluidized bed column and werediscarded. Data are provided in Table 16.

TABLE 16 DATA FROM A FLUIDIZED BED COLUMN OPERATION Flow Rate Volume ofBroth (liters per processed Time (minutes) minute) (liters) Comments 09.8 0 Start 10 2.9 20 60 2.9 160 70 2.9 160 Added 90 liters of harvestrecovery buffer to bioreactor 100 2.9 253 FinishG. Microfiltration of the Microcarrier-clarified Broth—Lysing theInfected Cells and BENZONASE™ Endonuclease Treatment

The starting material for the microfiltration process was the broth fromthe fluidized bed column that was clarified of the microcarriers andcontained cells and virus. During the microfiltration step, the cellswere lysed due to the shear rate used, the broth was clarified of debrislarger than 0.65 microns and the residual nucleic acids from the lysedcells was digested by BENZONASE™ endonuclease (e.g., 0.5 million unitsper 200 L batch), an enzymatic preparation.

The microfiltration unit was a PROSTAK system (Millipore). It containeda DURAPORE, 0.65 micron pore size, hydrophilic, membrane (catalog numberSK2P446EO) with a surface area of 54 square feet. The feed and retentatelines of the PROSTAK filter unit were connected to the microfiltrationrecirculation vessel which contained the microcarrier-clarified brothfrom the fluidized bed column. A line used to feed the harvest recoverybuffer into the microfiltration recirculation vessel was connected. Thepermeate line from the PROSTAK unit was connected to the ultrafiltrationrecirculation vessel. The temperature of the broth was maintained in therange of 25-35° C. When the broth feeding into the PROSTAK unit wasreduced to a volume of 10 to 30 liters in the microfiltrationrecirculation vessel, 50 liters of the harvest recovery buffer was addedto the vessel and the microfiltration continued. This step was repeatedonce. The microfiltration continued until the volume in themicrofiltration recirculation vessel was reduced to 10 to 30 liters. Atthis time, 0.5 million units of BENZONASE™ endonuclease was added to theclarified both in the ultrafiltration recirculation vessel. The contentsof the vessel were mixed well and the broth was held for two hoursbefore the ultrafiltration was started. Data are provided in Table 17.

TABLE 17 DATA FROM A MICROFILTRATION OPERATION Feed Permeate InletRetentate Permeate Broth volume flowrate flowrate pressure pressurepressure processed Time (m³/hr) (m³/hr) (mbar) (mbar) (mbar) (liters)Comments 0 9.6 0.17 1190 273 678 2 4 9.6 0.16 1206 278 664 12 20 9.60.16 1225 258 571 56 36 9.6 0.15 1230 249 551 98 50 9.6 0.16 1225 244532 138 63 0 176 Stop and add 50 liters of buffer 71 9.6 0.18 1250 268541 176 Start after buffer addition 81 9.6 0.18 1250 249 532 207 91 0 0234 Stop and add 50 liters of buffer 96 9.6 0.18 1250 253 522 237 Startafter buffer addition 102 9.6 0.18 1264 249 527 258 112 9.7 0.17 2284249 524 286 118 299 FinishH. Ultrafiltration of Broth to Concentrate the Virus with Diafiltrationto Perform Buffer Exchange

The starting material for the ultrafiltration process in theultrafiltration recirculation vessel was the BENZONASE™endonuclease-treated, clarified broth from the microfiltration permeate.The ultrafiltration unit was a PELLICON system (Millipore). It containeda 1 million nominal molecular weight cut-off, PELLICON II-regeneratedcellulose membrane (catalog number P2C01MC05) with a surface area of 40square feet. The feed and retentate lines of the PELLICON unit wereconnected to the ultrafiltration recirculation vessel. Theultrafiltration permeate line was connected to a waste vessel. A vesselcontaining the harvest recovery buffer (50 mM Tris base, 150 mM sodiumchloride, 2 mM magnesium chloride hexahydrate, and 2% sucrose) wasconnected to the ultrafiltration recirculation vessel. When theultrafiltration retentate volume reached 5 to 10 liters, an addition of15 liters of the harvest recovery buffer was made and theultrafiltration was continued. This step was repeated once. Theultrafiltration was continued until the retentate volume was less than 5to 10 liters. The retentate from the ultrafiltration contained theconcentrated virus. The retentate was collected from the PELLICON unit.A flush of 3 to 6 liters of the harvest recovery buffer was used tocollect all of the material from PELLICON unit. This flushed materialwas added to the ultrafilter retentate broth. This was filtered througha Millipore, DURAPORE, 0.45 micron pore size filter (catalog numberCVHL71PP3) into a sterile bag. The material in the sterile bag wasstored frozen at 80° C. Data are provided in Table 18.

TABLE 18 DATA FROM AN ULTRAFILTRATION OPERATION Flow rate (liters InletRetentate Permeate Time per pressure pressure pressure (minutes) minute)(bar) (bar) (bar) Comments 0 16.5 0.9 3.1 0.4 Start 5 16.5 0.9 3.1 0.439 17.3 0.9 3.1 0.4 86 17.3 0.9 3.1 0.4 Stop and added buffer 93 17.30.9 3.0 0.4 Start after buffer addition 100 Stop and added buffer 10715.7 0.9 3.0 0.4 Start after buffer addition 119 15.7 0.8 3.0 0.4Flushed unit - finishI. General Comments

All 293 cell cultures in T75, T500 and CELL FACTORY™ tissue cultureflasks were cultivated in Medium 1 in an incubator at 37° C., 100%humidity and a 5% CO₂ atmosphere. All open operations were performedaseptically under a biosafety (laminarflow) hood. Medium fills andadditions were performed through a 0.2 micron pore size, PALL ULTIPORN66, in-line filter installed on the feed port of the bioreactor whichwas steam sterilized for 30 minutes at 121° C. All other additions tothe bioreactors were performed each using a sterile Erlenmeyer flaskwith PHARMED™ tubing that was aseptically connected between thebioreactor and the addition flask by a tubing welder. All buffers usedin the recovery process were filtered through a 0.2 micron pore size,PALL ULTIPOR N66, inline filter (SLK7002NFP) installed on a port of thereceiving vessel. Note that for the microfiltration operations either ahydrophilic or hydrophobic membrane can be used.

All publications and patent applications cited herein are incorporatedby reference in their entirety to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Modifications and variations of this invention will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is not to be construedas limited thereby.

1. A system for separating cells from microcarriers on which the cellshave been cultivated, the system comprising: (a) a bioreactor in whichthe cells were cultivated on the microcarriers; (b) a flow path from thebioreactor to a separation device; (c) a separation device comprising:(i) an inlet situated at the bottom of the device; (ii) a single columnhaving a cylindrical upper section and a conical lower section, whereinthe walls of the cone are at an angle which allows the microcarriersthat settle on the walls to move downward to the inlet; (iii) an outlet,for the collection of cells and the aqueous solution, situated at thetop of the device; and (iv) a mesh screen located on the upper end ofthe column; wherein the microcarriers are retained in suspension by anupward flow in the separation device and are retained in the separationdevice by a mesh screen, and the cells and aqueous solution arecollected through the outlet; and (d) a pump, wherein the pump directsthe flow of the aqueous solution from the bioreactor to the outlet. 2.The system of claim 1, further comprising a microfilter.
 3. The systemof claim 2, further comprising an ultrafilter.
 4. The system of claim 1,wherein the upward flow is created by pumping the aqueous solutionthrough the inlet.
 5. The system of claim 1, wherein the lower sectionof the column comprises about 20 to 50% of the volume of the column. 6.The system of claim 5, wherein the angle of the lower section is about15 to about 45 degrees.
 7. The separation device according to claim 1.